Method of Delivery of Nucleic Acids to Peripheral Neurons

ABSTRACT

The invention provides for methods for delivering a nucleic acid into a peripheral neuron by identifying a target neuron in a dorsal root ganglion and intrathecally delivering a vector comprising the nucleic acid to the dorsal root ganglion neuron. The nucleic acid may encode a neurotrophic factor that may be used to treat a peripheral neuropathy or, in conjunction with a nerve guide conduit, to treat a transected peripheral nerve.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fordelivering nucleic acids to peripheral neurons.

BACKGROUND OF THE INVENTION

Nerve regeneration is a complex biological process. The degenerativeprocesses following damage to nerves in the central and peripheralnervous system are similar in some respects but different in others. Oneof the largest differences is that peripheral nerves have a much greatercapacity to regenerate their axons following nerve injury (Fenrich, K.et al. 2004, Can J Neurol Sci. 31(2):142).

Schmidt and Leach (2003, Annu. Rev. Biomed. Eng. 5:293) have recentlyreviewed a number of ways of treating nerve injuries. Current treatmentsfor injury-induced nerve defects typically rely on donor tissuesobtained from the patient. This has raised the issues of loss offunction at the donor sites, formation of potentially painful neuromas,structural differences between donor and recipient nerves and a shortageof graft material for extensive repair. To circumvent these problems,synthetic nerve guide conduits (NGCs) have been developed to bridge thenerve gaps by securing the severed nerve stumps into the two ends of theconduit (U.S. Pat. No. 5,019,087). A number of devices, such as, forexample, Integra Neurosciences Type I collage tube and SaluMedica'sSaluBridge™ Nerve Cuff have been approved by the US Food and DrugAgency. These devices, however, are reserved for treatment of relativelyshort nerve defects, and in most cases the synthetic conduits do notfunction as well as nerve autografts (Schmidt & Leach 2003, Annu. Rev.Biomed. Eng. 5:293).

Several tissue-engineering approaches have been proposed to enhance theperformance of NGCs, which include delivering neurotrophic factorswithin hollow tubes. Filling silicone NGCs with dialyzed plasma resultedin a three to fivefold increase in functional restitution at eight weekscompared to NGCs filled with phosphate buffered saline (Williams et al.1987, J. Comparative Neurology 264:284). Alternatively, neurotrophicfactors such as nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), fibroblast growth factor (FGF), glial growth factor (GFG)and ciliary neurotrophic factor (CNTF) delivered within a conduit maysignificantly increase the morphological and/or functional recovery oftransected and repaired nerves.

NGF is the first and best-characterized nerve-derived factor and acts ona relatively limited variety of neuronal populations, includingsympathetic, subpopulations of sensory neurons of peripheral nervoussystem and striatial and septal cholinergic neurons in the brain(Terenghi, G. 1999, J. Anat 194:1-14). In normal circumstances, NGF ispresent at a very low concentration but rapidly increases in theexperimental nerve injury animal model. NGF is produced mainly by thetarget tissue and Schwann cells in the distal stump of damaged nervesand then transported in a retrograde manner to the cell soma beforeacting on receptors on neurons and producing the neurotrophic effects.In peripheral neuropathies, such transport within diseased nerves may beaffected, being reduced or totally blocked. While delivery of NGFpromotes nerve regeneration within conduits at an early stage, thepromoting effect may not last after one month, probably due to the rapiddecline of NGF concentrations in the conduit caused by the degradationin aqueous media at 37° C., leakage from the conduit and/or dilution byentering fluids. Furthermore, the timing of the introduction ofneurotrophic factors into NGCs has a significant influence on thehealing or regenerative processes: introducing various agents too earlyor too late may inhibit the regenerative process (U.S. Pat. No.5,584,885).

The cell bodies of sensory neurons are located in dorsal root ganglia(DRG), nodules at the distal end of the dorsal root of each spinalnerve. Within the dural sheath and surrounded by the cerebral spinalfluid (CSF), dorsal and ventral nerve roots leave through theintervertebral foramen, where the dorsal root forms the dorsal rootganglion and thereafter joins the ventral root to form the spinal nerveroot. Morphologically, a somatosensory neuron in DRG has a unipolarstructure, with a connection to the central nervous system (CNS) by along ascending axon within the spinal cord, and to the peripheralnervous system (PNS) by a second axon branch descending through thespinal nerve root and further out into a peripheral nerve. Functionally,DRG neurons are heterogeneous, signaling receptor-transduced stimuli ofdiverse sensory modalities that range from touch, temperature, pain toproprioception.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method of delivering a nucleicacid into a neuronal cell in the peripheral nervous system of a hostcomprising the step of identifying a target neuronal cell in a dorsalroot ganglion and administering a vector comprising the nucleic acidinto a site in the cerebrospinal fluid of the host wherein the site issufficiently proximal to the dorsal root ganglion to deliver the nucleicacid into the cell body of the target neuronal cell.

In another aspect the invention provides a method of treating aperipheral neuropathy in a host, the method comprising identifying atarget neuronal cell in a dorsal root ganglion affected by theneuropathy, and administering a vector comprising a therapeutic nucleicacid into a site in the cerebrospinal fluid of the host wherein the siteis sufficiently proximal to the dorsal root ganglion to deliver thenucleic acid into the cell body of the target neuronal cell.

In another aspect the invention provides a method of treating atransected peripheral nerve having a proximal and a distal stump in ahost, the method comprising intrathecally administering a vectorcomprising a therapeutic nucleic acid to a dorsal root ganglion neuronalcell wherein the distal and proximal stumps are secured to a nerve guideconduit.

In another aspect the invention provides a use of a vector comprising anucleic acid to deliver the nucleic acid into a target neuronal cell ina dorsal root ganglion wherein said delivery is effected from a site ofadministration of the vector in the cerebrospinal fluid which issufficiently proximal to the dorsal root ganglion to deliver the nucleicacid into the cell body of the target neuronal cell.

In yet another aspect the invention provides a use of a vectorcomprising a therapeutic nucleic acid to treat a peripheral neuropathyin a host wherein the nucleic acid is delivered into a target neuronalcell in a dorsal root ganglion affected by the neuropathy from a site ofadministration of the vector in the cerebrospinal fluid which issufficiently proximal to the dorsal root ganglion to deliver the nucleicacid into the cell body of the target neuronal cell.

In still yet another aspect, the invention provides a use of a vectorcomprising a therapeutic nucleic acid to treat a transected peripheralnerve having a proximal stump and a distal stump in a host wherein theproximal and distal stumps are secured to a nerve guide conduit andwherein the nucleic acid is delivered from a site of administration inthe cerebrospinal fluid to a dorsal root ganglion neuronal cell.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention:

FIG. 1 shows confocal scanning microscope images of Cy3 labeledbaculoviruses or PEI/DNA complexes in rat DRG at 2 days post-injection.The DRG neurons are stained with FITC-labeled NeuN (green). Cy3 labeledbaculoviruses and PEI complexes (red) are found mainly in the cytoplasmof cells and well co-localized with NeuN signals (arrows);

FIG. 2 shows transfection of DRG cells with PEI/DNA or baculovirusvectors encoding a firefly luciferase (Luc) gene one day (top panel) orthree days (lower panel) after intrathecal injection. The 540 bp PCRfragment corresponds to a portion of the firefly luciferase gene.

FIG. 3 shows luciferase expression from four different vectors with aCMV E/P promoter in DRG. Luciferase activities were measured at 2 dayspost-injection and are expressed in relative light units (RLU) permilligram of protein (FIG. 3A) or per tissue (FIG. 3B). FIG. 3C depictsthe time-dependent change of luciferase activity, expressed in RLU permilligram of protein, in DRG after intrathecal injection of PEI/DNAcomplexes.

FIG. 4 shows luciferase expression from four different vectors with aCMV E/PDGF promoter in the spinal cord and the DRG. Luciferaseactivities were measured at 2 days post-injection and are expressed inrelative light units (RLU) per tissue (FIG. 4A) or per milligram ofprotein (FIG. 4B).

FIG. 5 shows confocal scanning microscope images of luciferaseexpression in neurons in DRG. Frozen sections of rat DRG collected 2days after injection of PEI/DNA complexes were used for doubleimmunostaining against luciferase to show transfected cells and againstNeuN to show neurons. Most of the cells are well co-localized (NeuN+Luc, arrows), with a few of luciferase positive cells being not labeledby NeuN (arrow heads).

FIG. 6 shows NGF concentrations after in vitro (A, B) and in vivo (C)gene transfection. The concentrations of NGF in COS7 cell cultures areexpressed per ml of culture medium (FIG. 6A) or per mg protein of celllysate (FIG. 6B). The cells were collected 24 hours after in vitrotransfection. DRG were collected 3 and 7 days after intrathecalinjection of PEI complexes containing pcDNA3-NGF or a control plasmidpcDNA3-luc (FIG. 6C)

FIG. 7 shows morphometric analysis of nerve regeneration within NGCs 4weeks after intrathecal injection. The samples from the PEI/pcDNA-NGFtransfection group show larger diameter of fibers and a lower G ratio ascompared with those from the control group (p<0.01). There is nosignificant difference between the two groups in terms of fiberpopulation and density.

FIG. 8 shows electron microscope morphology of regenerated nerve fibersfrom the NGF transfected (B) and control (A) groups. Samples werecollected 4 weeks after operation. MA: myelinated axons. Note largeraxons with thicker myelin in the NGF group. The original magnification×10000.

DETAILED DESCRIPTION

The inventors have surprisingly discovered that the administration ofnucleic acid vectors into cerebrospinal fluid surrounding the spinalchord is an effective way of delivering exogenous nucleic acids into thesoma of neurons in dorsal root ganglia. More specifically, nucleic acidvectors encoding NGF intrathecally injected into cerebrospinal fluidwere to shown to positively influence the regeneration of a transectedsciatic nerve within a nerve guide conduit.

The terms “neuronal cell” and “neuron”, which are use interchangeablyherein, in accordance with the usual meaning in the art, refer to anyconducting cell of the nervous system, which typically includes the cellbody or soma, several dendrites and an axon. The terms include a singlecell as well as a plurality or population of cells unless the contextclearly indicates otherwise.

The intrathecal administration of recombinant vectors into thecerebrospinal fluid proximal to a dorsal root ganglion allows for thetransfection of dorsal root ganglion neuronal cells in a manner that maybe independent of axonal transport and may be accomplished by relativelyminimally invasive techniques. Following peripheral nerve injury, cellsand tissues surrounding the injury site, including Schwann cells,normally secrete factors that are taken up by the axon of the injured ordamaged neuron. These factors are generally transported to the soma ofthe injured nerve by axonal transport, where they interact withreceptors and exert biological effects. A number of viral-based genetherapy system, including herpes and poliovirus based therapies, haveexploited this axonal transport to deliver therapeutic genes tootherwise inaccessible neurons, including those within the centralnervous system. As would be appreciated by a person skilled in the art,these approaches may provide less than optimal gene delivery incircumstances where axonal transport is compromised, such as, forexample, in certain neuropathies, such as, for example, diabeticneuropathy and peripheral nerve injuries caused by trauma, compressionor transection.

By using intrathecal injection to target DRG neurons, nucleic acidvectors encoding exogenous genes under the control of an appropriatepromoter may be delivered to the soma of cells in the DRG incircumstances where peripheral axonal transport is compromised orabolished. Therefore, a nucleic acid may be delivered to a neuronal cellbody in the peripheral nervous system by the administration of a vectorcomprising the nucleic acid into cerebrospinal fluid. As will beunderstood, the site of injection in the cerebrospinal fluid will dependon the specific target neuronal cells of a dorsal root ganglia intowhich the delivery of a nucleic acid is desired. More specifically, thesite of administration may be selected to be sufficiently proximal tothe target DRG neuronal cells to deliver the nucleic acid into the cellbody of the targeted cells, meaning, the delivery can be mediated oreffected independently of axonal transport. Therefore, nucleic acids maybe delivered to target cells independent of axonal transport. A personskilled in the art would appreciate, from the segmental architecture ofthe peripheral nervous system, that the site of administration willdepend on which DRG nerve cells are targeted for transfection. Forexample, in order to deliver a transgenic gene product to peripheralnerves extending into in the index finger of a subject, the vector ispreferably intrathecally administered into the CSF surrounding or nearthe C6 vertebrae of the subject. Appropriate sites of injection may bedetermined by reference common anatomy texts, such as, for example,Introduction to Human Anatomy 6^(th) edition, Francis, C. V. MosbyCompany, 1973.

In a specific embodiment, the vector is administered by lumbarinjection. Lumbar injection is considered safe and poses little dangerof injuring the spinal cord and nerve roots, as cerebrospinal fluid(CSF) in the relatively wide subarachnoid space at the level of thecauda equina allows a certain degree of mobility of the nerve roots inresponse to needle puncture. This method would therefore permit safemultiple administrations for gene delivery to lumbar DRGs. Lumbarpuncture has been clinically used as an access method for spinalanesthesia and for introduction of therapeutic or diagnosis agents.

An intrathecally administered vector comprising a nucleic acid sequencemay therefore be used to direct the expression of any exogenous nucleicacid in neuronal cells located in dorsal root ganglia to providetherapeutic products to peripheral neurons following traumatic injury ordisease or to study gene expression in peripheral neurons.

In different embodiments, the vector is a viral vector. “Viral vector”refers to recombinant viruses engineered to effect the introduction ofexogenous nucleic acids into cells. Viral vectors include, for example,retroviruses, adenoviruses, adeno-associated viruses (AAV),baculoviruses, vaccinia viruses, herpes viruses, alphavirsus vectors,alphavirus replicons and lentivirus vectors.

The delivery of nucleic acids into a cell by a viral vector may requirespecific interactions between molecules on the outer surface of theviral envelope and molecules on the cell to be transfected, for example,such as those between the glycoprotein D and the cell surface receptors,herpesvirus entry mediator A, or nectin-1 (Krummenacher et al. 2003,Journal of Virology 77(16): 8985). Alternatively, the viral genedelivery system may involve non-specific interactions, for example, suchas the baculoviral infection of mammalian cells. Baculoviruses display abroad tropism for mammalian cells and viral entry may be mediated byelectrostatic interactions which may not be cell-specific (Sarkis et al.2000, Proc. Nat Acad. Sci. 97:14638). Depending on the nature of thetarget cell sought to be transfected, a person skilled in the art canreadily determine which of the viral gene-delivery systems may be themost appropriate

In specific embodiments, the viral vector may be a baculovirus vector oran AAV vector. Baculovirus vectors have recently been viewed as a newgeneration of gene therapy vehicles, due to their broad tropism in bothproliferating and non-proliferating quiescent mammalian cells, the lackof replication in vertebrate cells and little to no microscopicallyobservable cytotoxcity (Ghosh et al., 2002, Mol Ther 6:5; Kost &Condreay, 2002, Trends Biotechnol 20:173). Baculovirus vectors, such as,for example, those derived from Autographa Californica MulticapsidNucleopolyhedrovirus (AcMNPV), may be well suited for gene therapy ofnon-dividing cells because they are episomal and their promoters aresilent in mammalian cells, making them non replicative in human cells(Sarkis et al. 2000, Proc. Nat. Acad. Sci. 97: 14638). Baculovirusvectors have been shown to transfect brain cells when directly injectedin vivo (Sarkis et al. 2000, Proc. Nat. Acad. Sci 97: 14638;Tani et al.2003, Journal of Virology 77(18):9799). Further, relative to adenoviralvectors, baculovirus vectors may elicit much less of a microgliaresponse (Lehtolainen et al. 2002, Gene Therapy 9:1693).

The possibility of using baculovirus vectors for gene transfection inthe nervous system has been investigated in two studies. An initialreport described the efficient transduction of neural cells in vitro andin vivo (Sarkis et al., 2000, Proc. Nat. Acad. Sci 97: 14638). Inprimary cell cultures of human embryonic brains, neuroepithelial,neuroblastic, and glial cells could be infected, although in vivostudies using adult nude mice demonstrated that mainly astrocytes andonly a few neurons were transduced. The second study examined thecell-type specificity of baculovirus-mediated gene expression in thebrain and identified cuboidal epithelial cells of the choroids plexus asthe main target, with modest gene expression in endothelial cells andvery limited or no expression in other types of brain cells, includingneurons and astrocytes (Lehtolainen et al., 2002, Gene Therapy 9:1693).

The inventors have shown for the first time that baculoviruses arecapable of infecting sensory neurons in DRG by intrathecal injection.

A person skilled in the art would readily appreciate how to constructbaculoviral vectors for use in the invention. Recombinant baculovirusvectors may be constructed according to instructions accompanyingcommercial baculovirus expression systems, for example, the Bac-to-Bac®Expression system (Invitrogen). Recombinant baculoviral vectors may bemodified by molecular biological techniques, including PCR-basedtechniques and other cloning techniques, as will be known to a skilledperson and described, for example, in Sambrook et al., Molecular CloningA Laboratory Manual (3^(rd) ed.), Cold Spring Harbour Press.

Viral vectors may be engineered to contain increased levels of the viralenvelope glycoprotein gp64. Although the mechanism of action of gp64 onviral entry into mammalian cells is unknown, viral vectors withincreased levels of pg64 have enhanced levels of transduction (Tani etal. 2001, Virology 279: 343). Recombinant viral vectors may also bemodified by incorporating foreign envelope proteins into the envelope ofthe viral virion. For example, increased neural infection efficiency maybe achieved by pseudotyping (Sarkis et al. 2000, Proc. Nat. Acad. Sci.97: 14638) rabies virus glycoprotein (RVG) or vesicular stomatitis virusG protein (VSVG) (Tani et al. 2003, Journal of Virology 77(18): 9799),herpes envelope glycoprotein or envelope proteins derived from α- orrhabdovirus (Ghosh et al. 2002, Molecular Therapy 6(1):5) into theenvelope of the viral virion. RVG is known to use the nicotinicacetylcholine receptor and the low affinity nerve growth factor receptorfor viral entry, and a RVG-modified baculovirus has been shown to have10-5000 fold higher efficiency of neural cell transfection than theunmodified baculovirus (Tani et al. 2003, Journal of Virology 77(18):9799). Alternatively, the cell specificity of viral infection may beincreased by incorporating antibodies directed against cell-specificprotein receptors into the viral envelope.

To minimize or avoid any possibility for inactivation by serumcomplement (Tani et al. 2003, Journal of Virology, 77(18):9799),recombinant viruses may be modified to increase their resistance to thecomplement system, including, for example, by incorporating humandecay-accelerating factor into a viral envelope (Hüser et al., 2001,Nature Biotechnology 19:451).

In other embodiments, the vector is a non-viral vector. “Non-viralvectors” refers to systems other than viral vectors that may be used tointroduce exogenous nucleic acids, for example plasmids, into a cell.Non-viral vectors include, but are not limited to polymer-based,peptide-based and lipid-based vectors. Many non-viral vectors arecommercially available, such as, for instance PEI 25K (Sigma-Aldrich,St. Louis, Mo.) Lipofectamine™ 2000 (Invitrogen, Carlsbad Calif.).Complexes of these vectors and nucleic acids may be prepared accordingto commercial instructions, or by following protocols known to a personskilled in the art, such as, for example, Boussif et al. (1995, Proc.Nat. Acad. Sci. 92:7297).

Generally, non-viral gene-delivery systems rely on the direct deliveryof the target nucleic acid or on nonspecific internalization methods.Non-viral gene delivery systems and methods for their transfection wouldbe known to a person skilled in the art, and include, for example, nakedplasmids, DEAE-dextran, calcium phosphate co-precipitation,microinjection, liposome-mediated transfection, cationic lipids, andpolycationic polymers. As would further be appreciated by a personskilled in the art, some of these methods, such as, for example,microinjection, liposome-mediated transfection, polycationic polymers,are capable of transfecting cells both in vivo and in vitro. Thesenon-viral vectors may be modified to enhance nerve-specifictransfection, for example by linking the vector to one or more ligandsthat may specifically or preferentially bind to neuronal cells. Forexample, nerve-specific transfection of polylysine/DNA complexes may beobtained by covalently linking the nontoxic fragment C of tetanus toxinto polylysine (Knight et al. 1999, Eur J. Biochem 259: 762-769).

Non-viral vectors containing DNA with bacterial sequences often haveincreased palindromic CpG sequences relative to eukaryotes, and theseforeign CpG sequences may serve as strong immunostimulatory agents invertebrates. Reducing CpG content therefore may be advantageous and mayalso enhance protein expression as CpG sequences may be methylated ineukaryotic hosts, which can result in the transcriptional silencing(Chevalier-Mariette et al. 2003, Genome Biology 4:R53). In someembodiments, the CpG content of the DNA of non-viral DNA-based vectorsis reduced. Methylation of cytosine residues within a CMVpromoter-enhancer (CMV P/E) of a first generation adenovirus, includingat CpG sites, has been shown to be the major mechanism for decreasedtransgene expressions (Brooks et al. 2004, J. Gene Med. 6:395). A personskilled in the art would readily appreciate that the CpG dinucleotidecontent of a vector may be reduced using standard molecular biologytechniques, such as oligonucleotide or PCR-based mutagenesis asdescribed, for example, in Chevalier-Mariette et al. 2003, GenomeBiology 4:R53.

In some embodiments, the non-viral vector is a polyethyleneimine/DNAcomplex (PEI/DNA). Polycationic PEI has a high transfection efficiencyboth in vitro and in vivo (Boussif et al. 1995, Proc. Nat. Acad. Sci.92:7297). Preferably, the DNA in PEI/DNA is plasmid DNA. In PEI/DNAcomplexes, the ratio of PEI nitrogen to DNA phosphate is preferably 6 to30, more preferably 6 to 20 and most preferably 6 to 15 (Boussif et al.1995, Proc. Nat. Acad. Sci. 92:7297). A person skilled in the art canreadily prepare PEI/DNA, for example, by following establishedcommercial protocols. Once in the nervous system, PEI may mediate DNAtransfection in terminally differentiated non-dividing neurons (Boussifet al. 1995, Proc. Nat. Acad. Sci. 92:7297). After direct braininjection, PEI/DNA complexes can provide transgene expression levelshigher than those obtained with HIV-derived vectors and within the samerange as that achieved with adenoviral vectors. The PEI in a PEI/DNAcomplex may have an average molecular weight of 800 kD, 50 kD, or morepreferably 25 kD (Abdallah et al. 1996, Hum Gene Ther. 7(16):1947). PEImay also be covalently modified with other polymers, such as for examplepolyethylene glycol to reduce the cytotoxicity of the PEI/DNA complex.(Shi et al. 2003, Gene Therapy 10, 1179).

In different embodiments, the vector comprises a promoter operablylinked to a coding nucleic acid sequence. The promoter may be a strongviral promoter such as CMV or a neuronal-specific promoter. Specificgene expression in a selected cell type can be achieved by using acell-specific promoter.

Neuron-specific promoters may be any nucleotide sequence that functionsto activate transcription of operably linked sequences within neurons orneuronal cells and substantially not in other cells types. A promoterdoes not substantially activate transcription if the levels oftranscription of operably linked sequences in any of those cell typesare sufficiently low so as not to affect the physiological functioningof the cell.

Neuron-specific promoters may include promoters for neuronal genes suchas Synapsin I, Neuron-specific enolase, Neurofilament-L and NeuropeptideY and promoters specific for particular types of neuronal cells. Forexample, tyrosine hydroxylase gene promoter (4.8 kb 5′ UTR) is specificfor catecholaminergic and the CNS neurons, dopamine-b-hydroxylase genepromoter is specific for adrenergic and noradrenegic neurons and L7Purkinje cell protein promoter is specific for retinal rod bipolarneurons. 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Neuron-specific expression in vivo bydefined transcription regulatory elements of the GnRH gene.Endocrinology 2002; 143:1404-1412; Wolfe A, Kim H H, Tobet S, Stafford DE, Radovick S. Identification of a discrete promoter region of the humanGnRH gene that is sufficient for directing neuron-specific expression: arole for POU homeodomain transcription factors. Mol Endocrinol 2002;16:435-449; Makinae K, Kobayashi T, Kobayashi T, Shinkawa H, Sakagami H,Kondo H, Tashiro F, Miyazaki J, Obata K, Tamura S, Yanagawa Y. Structureof the mouse glutamate decarboxylase 65 gene and its promoter:preferential expression of its promoter in the GABAergic neurons oftransgenic mice. J Neurochem 2000; 75:1429-14371; Hitoshi S, Kusunoki S,Kanazawa I, Tsuji S. Dorsal root ganglia neuron-specific promoteractivity of the rabbit beta-galactoside alpha1,2-fucosyltransferasegene. J Biol Chem 1999; 274:389-396; Roztocil T, Matter-Sadzinski L,Gomez M, Ballivet M, Matter J M. Functional properties of the neuronalnicotinic acetylcholine receptor beta3 promoter in the developingcentral nervous system. J Biol Chem 1998; 273:15131-15137; Luscher B,Hauselmann R, Leitgeb S, Rulicke T, Fritschy J M. Neuronalsubtype-specific expression directed by the GABA(A) receptor deltasubunit gene promoter/upstream region in transgenic mice and in culturedcells. Brain Res Mol Brain Res 1997; 51:197-211; Zambrano N, De RenzisS, Minopoli. G, Faraonio R, Donini V, Scaloni A, Cimino F, Russo T.DNA-binding protein Pur alpha and transcription factor YY1 function astranscription activators of the neuron-specific FE65 gene promoter.Biochem J 1997; 328:293-300; Kim D S, Jung H H, Park S H, Chin H.Isolation and characterization of the 5′-upstream region of the humanN-type calcium channel alpha1B subunit gene. Chromosomal localizationand promoter analysis. J Biol Chem 1997; 272:5098-5104 and Liu D,Fischer I. Two alternative promoters direct neuron-specific expressionof the rat microtubule-associated protein 1B gene. J Neurosci 1996;16:5026-5036. Other neuron-specific promoters will be known to personsskilled in the art.

A neuron-specific promoter comprises at least one nucleotide sequencecapable of activating neuronal cell specific expression of operablylinked sequences and in some embodiments the nucleotide sequence willretain the minimum binding site(s) for transcription factor(s) requiredfor the sequence to act as a promoter. In some embodiments, the vectorcomprises multiple copies of the same sequence or two or more differentnucleotide sequences each of which is effective to activatetranscriptional activity. For various promoters which may be used,transcription factor binding sites may be known or identified by one ofordinary skill using methods known in the art as described above.Suitable promoter/enhancer constructs may be readily determined bystandard expression assays.

Platelet-derived growth factor β-chain (PDGF β) promoter (Sasahara M,Fries J W, Raines E W, Gown A M, Westrum L E, Frosch M P, Bonthron D T,Ross R, Collins T. PDGF β-chain in neurons of the central nervoussystem, posterior pituitary, and in a transgenic model. Cell 1991;64:217-227) has been shown to be specific for neuronal cells, includingdopaminergic neurons and in one embodiment, the neuron-specific promoteris PDGF β promoter. In a specific embodiment, the neuron-specificpromoter is human PDGF β promoter.

The transcriptional activity of a promoter in some instances may beweak, providing a less than ideal level of expression of therapeuticgene sequences. In various embodiments, the promoter may be operablylinked to an enhancer. As would be understood by a skilled person, an“enhancer” is any nucleotide sequence capable of increasing thetranscriptional activity of an operably linked promoter and, in the caseof a neuron-specific promoter, of selectively increasing thetranscriptional activity of the promoter in neuronal cells. A number ofenhancers are known and a person skilled in the art would also know howto screen for novel enhancer sequences, for instance, by screeningnucleotide sequences capable of increasing the transcription of areporter gene, for instance, through functional mapping.

A first nucleic acid sequence is operably linked with a second nucleicacid sequence when the sequences are placed in a functionalrelationship. For example, a coding sequence is operably linked to apromoter if the promoter activates the transcription of the codingsequence. Similarly, a promoter and an enhancer are operably linked whenthe enhancer increases the transcription of operably linked sequences.Enhancers may function when separated from promoters and as such, anenhancer may be operably linked to a promoter even though it is notcontiguous to the promoter. Generally, however, operably linkedsequences are contiguous.

In different embodiments, the enhancer may be a heterologous enhancer,meaning a nucleotide sequence which is not naturally operably linked toa promoter and which, when so operably linked, increases thetranscriptional activity of the promoter. Reference to increasing thetranscriptional activity is meant to refer to any detectable increase inthe level of transcription of an operably linked sequence compared tothe level of the transcription observed with a promoter alone, as may bedetected in standard transcriptional assays, including those using areporter gene construct.

The enhancer may be a known strong viral enhancer element such as Roussarcoma virus (RSV) promoter (Gorman et al 1982. Proc. Nat. Acad. Sci.79:6777-6781), SV40 promoter (Ghosh et al. 1981, Proc. Nat. Acad. Sci.78:100), CMV enhancer or promoter including CMV immediate early (IE)gene enhancer (CMVIE enhancer) (Boshart et al 1985, Cell 41:521; Niwa etal. 1991, Gene 108:193; see also U.S. Pat. Nos. 5,849,522 and5,168,062).

In one embodiment of the present invention, a CMV enhancer is operablylinked upstream to PDGF β promoter. In a further embodiment, CMVEenhancer is operably linked upstream to PDGF β promoter and the twosequences are contiguous. In another embodiment, a CMV enhancer isoperably linked to a CMV promoter (CMV E/P).

Further examples of neuron-specific promoters that may be operablylinked to enhancers, including allelic variants and derivatives of knownpromoter and enhancer sequences, are described in U.S. application Ser.No. 10/407,009.

In various embodiments, such variants and derivatives may besubstantially homologous in that they hybridize to the known enhancerand promoter sequences under moderate or stringent conditions.Hybridization to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH. Stringenthybridization may, for example, be conducted in 5×SSC and 50% formamideat 42° C. and washed in a wash buffer consisting of 0.1×SSC at 65° C.Washes for stringent hybridization may, for example, be of at least 15minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105minutes or 120 minutes.

The degree of homology between sequences may also be expressed as apercentage of identity when the sequences are optimally aligned, meaningthe occurrence of exact matches between the sequences. Optimal alignmentof sequences for comparisons of identity may be conducted using avariety of algorithms, such as the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithmof Needleman and Wunsch, 1970, J. Mol Biol. 48:443, the search forsimilarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.USA 85: 2444, and the computerised implementations of these algorithms(such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, Madison, Wis., U.S.A.).Sequence alignment may also be carried out using the BLAST algorithm,described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using thepublished default settings). Software for performing BLAST analysis maybe available through the National Center for Biotechnology Information(through the internet at http://www.ncbi.nlm.nih.gov/). In variousembodiments, the variants and derivatives may be at least 50%, at least80%, at least 90% or at least 95% identical as determined using suchalgorithms.

In different embodiments, the vector comprises a gene encoding a markerprotein whose expression and cellular or subscellular localization maybereadily determined. “Marker protein” refers to a protein whose presenceor subscellular localization may be readily determined, such as a greenfluorescent protein (GFP) or any of its enhanced derivatives. Othermarker proteins would be known to a person skilled in the art. Indifferent embodiments, the gene may encode an enzyme whose expressionmay be readily determined by providing a specific substrate anddetecting the products of enzymatic turnover, such as, for example, byproviding luciferin to cell or cell lysates containing luciferase. Inother embodiments, the marker protein may be any protein whoseexpression may be detected immunologically, for example by providing alabeled antibody that specifically recognizes the marker protein. Theantibody is preferably a monoclonal antibody and may be directly orindirectly labeled according to methods known in the art, such as, forexample, labeling with a fluorescent dye and detecting expression of theprotein by fluorescence microscopy. Other immunological detectionmethods, including without limitation, immunogold staining,radiolabelling, colourometric enzymatic precipitation would be known toa person skilled in the art.

Preferably, the nucleic acid vector comprises a therapeutic gene or atherapeutic transgene whose expression produces a therapeutic product.The term “gene” is used in accordance with its usual definition, to meanan operatively linked group of nucleic acid sequences. As used herein,“therapeutic product” describes any product that effects a desiredresult, for example, treatment, prevention or amelioration of a disease.The therapeutic product may be a therapeutic protein, a therapeuticpeptide or a therapeutic RNA, such as, for example, a small interferingRNA (siRNA) or an anti-sense RNA.

In some embodiments, the therapeutic product is a neurotrophic factor,such as, for example a nerve growth factor. As used herein, “nervegrowth factor” refers to any factor that can promote nerve cell growthand/or nerve regeneration such as, for example, neurotrophins,neurotrophin receptors and neurotrophic factors. “Neurotrophin” refersto a protein family that can support neuronal survival and/or nerveregeneration. Neurotrophins include, for example, Nerve Growth Factor(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3),neurotrophin 4/5 (NT-4/5) as well as neurotrophin variants. A “variant”refers to a protein whose sequence differs from that of the naturallyoccurring protein by one or more amino acid substitutions, additions ordeletions but maintains some of the biological activity of the naturallyoccurring protein. As will be appreciated by a person skilled in theart, a variant may possess about 60%, 70%, 80% preferably 90%, or morepreferably greater than 95% homology with a naturally occurring protein.In specific embodiments, the therapeutic product is NGF. In a specificembodiment, the vector comprises a gene encoding NGF operatively coupledto CMV E/P.

“Neurotrophin receptor” refers to proteins that are able to bindneurotrophins. Neurotrophin receptors include, for example, thelow-affinity p75 receptors and high-affinity receptors such as, forexample, trkA, trkB, and trkC and their variants. “Neurotrophic factors”include, for example, ciliary neurotrophic factor (CNTF),hippocampus-derived neurotrophic factor (HDNF), leukemia inhibitoryfactor (LIF) (Yamamori et al. 1989, Science 246: 1412), acidic and basicfibroblast growth factor (aFGF, bFGF) and their variants.

“Neurotrophic factors” also include other factors that may enhanceneurite outgrowth in vitro and/or in vivo, that may potentiate axonalregeneration, or that may be effective in counteracting the negativeeffects of chronic axotomy on axonal regeneration, such as, for example,the immunophillin ligand FK506 (Lee et al. 2000, Muscle Nerve 23:633)and its variants.

In other embodiments, the therapeutic product is an anti-apoptoticfactor such as, for example, bcl-2 or bcl-XL. In adults, peripheralnerve damage is followed by 20 to 40% cell loss in DRG, which is likelyfrom apoptosis (Terenghi 1999, J. Anat. 194:1). Without being limited toany particular theory, it is believed that the delivery ofanti-apoptotic proteins to neuronal cells in the DRG may reduce or mayprevent this cell loss.

Methods for preparing recombinant vectors would be well-known to aperson skilled in the art, for example, those described in Sambrook etal. Molecular Cloning, A Laboratory Manual (3^(rd) ed) Cold SpringHarbour Laboratory Press (2001) and other laboratory manuals, and asdescribed in commercial instructions.

To aid in administration, the vectors may be formulated as an ingredientin a pharmaceutical composition. The compositions may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives and various compatible carriers or diluents. For all formsof delivery, the vectors may be formulated in a physiological saltsolution.

The proportion and identity of the pharmaceutically acceptable diluentis determined by chosen route of administration, compatibility with thevector and standard pharmaceutical practice. Generally, thepharmaceutical composition will be formulated with components that willnot significantly impair the biological activities of the vector.Suitable vehicles and diluents are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

Solutions of the vectors may be prepared in a physiologically suitablebuffer. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms, but thatwill not inactivate the vector. A person skilled in the art would knowhow to prepare suitable formulations. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences andin The United States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999.

In some embodiments, the vectors are administered to a vertebrate host.In a specific embodiment, the vectors are administered to a human host.

DRG have been considered as early and critical targets in several typesof peripheral neuropathies, including diabetic neuropathy, the mostprevalent form of peripheral neuropathies (Kishi et al 2002, Diabetes51:819; England & Asbury, 2004 Lancet 363:2151). Patients with diabeticneuropathy may display exclusive sensory and autonomic symptoms withoutobvious motor disorder. Pathological studies of human and experimentaldiabetic neuropathies have confirmed cell loss in DRG, which may haveresulted from the observed loss of mylenated fibers and axonal atrophy.Neurons in DRG are also directly involved in the pathophysiology ofperipheral nerve injuries caused by physical trauma, compression ortransection. Peripheral nerve transection may also cause biochemicalalterations in the expression of neuropeptides, cytokines andtranscription factors in the perikaryon of the surviving neurons in theaffected DRGs and atrophy in the proximal nerve stump (Stoll & Muller1999, Brain Pathol. 9(2):313). These pathological alterations willcertainly disturb the transmission of sensory signals from body partslike the skin, muscles and internal organs to the CNS. As a result,parts of the body may function inappropriately or not at all.

Expression of therapeutic genes, especially those encodingneurotrophins, in DRG prevents nerve degeneration in experimentalneuropathy (Glorioso et al. 2003, Curr Opin Mol Ther. 5:483). However,gene transfer to DRG is still challenging due to their anatomicfeatures. In previous studies, the transfer was achieved throughintramuscular or subcutaneous injection of viral vectors, mainly herpessimplex virus (HSV), that can be taken up by nerve terminals and thentransported in axoplasm to somas of neurons in DRG (Haase et al., 1998,J. Neurol Sci.160 Suppl.; Jackson et al., 2003, Virology 314:45; Goss etal. 2001, Gene Therapy 8:551). While being effective, this approachrelies on functioning cellular mechanisms including endocytosis at nerveterminals and retrograde axonal transport, which may already be damagedunder peripheral neuropathic conditions.

In another method not dependent on nerve terminal endocytosis andretrograde axonal transport, Glatzel et al. (2000, Proc. Nat. Acad. Sci.97:442) and Xu et al. (2003, Biomaterials 24:2405) have recently used amicroneurosurgical technique for direct injection of gene transfervectors into DRG, that resulted in strong expression of reporter genesalong sensory neural pathways. The injection procedure requires removinga piece of vertebra to gain access to DRG. It is also invasive to thetissues of DRG and not practical when a repeat injection scheme isrequired, such as, for example, for the long-term therapy of a chronicdisorder.

The minimally invasive delivery of vectors comprising genes encodingtherapeutic products to the cell bodies of neurons within dorsal rootganglia according to the invention therefore may be advantageously usedfor treating peripheral neuropathies to promote the growth and/orregenerate injured peripheral nerves. “Peripheral neuropathy”, as willbe understood by a person skilled in the art, refers to the loss ofperipheral neuronal cells or their function with or without obviousmotor disorder. Such cells are therefore target cells that are affectedby a peripheral neuropathy and that may be treated by expression oftherapeutic gene within the cells. Peripheral neuropathies may be causedby a disease or disorder or as the result of systemic illnesses. Manyneuropathies have well-defined causes such as, for example, diabetes,uremia, AIDS, Lyme disease or nutritional deficiencies. Other causes ofperipheral neuropathy include mechanical pressure such as compression orentrapment, direct trauma, penetrating injuries, contusions, fracture ordislocated bones; pressure involving the superficial nerves; intraneuralhemorrhage; exposure to cold or radiation or, rarely, certain medicinesor toxic substances; and vascular or collagen disorders such as, forexample, atherosclerosis, systemic lupus erythematosus, scleroderma,sarcoidosis, rheumatoid arthritis, and polyarteritis nodosa. Althoughthe causes of peripheral neuropathy are diverse, they generally producecommon symptoms including weakness, numbness, paresthesia (abnormalsensations such as burning, tickling, pricking or tingling) and pain inthe arms, hands, legs and/or feet. A large number of cases are ofunknown cause.

The vectors are administered preferably by intrathecal injection in anamount sufficient to achieve the desired result, for example, expressionof therapeutic gene in an effective amount in the target cells.

In some embodiments, the vector is administered by lumbar puncture.Lumbar puncture is a relatively routine and non-traumatic clinicalprocedure that poses minimal risk to the spinal chord. Generally, asolution comprising the vector is administered into the cerebrospinalfluid with a narrow needle, such as, for example a 26 gauge needle,connected to a syringe or microsyringe. In rats, the proper intrathecallocation of the needle may be confirmed by a slight tail movement,indicating the proper injection into the subarachnoid space. Afterinjection, the needle may remain in the intrathecal space for a periodof time, such as, for example, 2 minutes before being removed.

Effective amounts of vectors can be given repeatedly, depending upon theeffect of the initial treatment regimen. Administrations are typicallygiven periodically, while monitoring any response. It will be recognizedby a skilled person that lower or higher dosages may be given, accordingto the administration schedules and routes selected.

When administered to a human patient, for example, the vectors areadministered in an effective amount and for a sufficient time period toachieve a desired result. For example, the vectors may be administeredin quantities and dosages necessary to deliver a therapeutic gene, theproduct of which functions to alleviate, improve, mitigate, ameliorate,stabilize, prevent the spread of, slow or delay the progression of orcure a peripheral neuronal neuropathy.

The effective amount to be administered to a patient can vary dependingon many factors such as, among other things, the pharmacodynamicproperties of the therapeutic gene product, the mode of administration,the age, health and weight of the subject, the nature and extent of thedisorder or disease state, the frequency of the treatment and the typeof concurrent treatment, if any. In embodiments employing viral vectors,the effective amount may also depend on the virulence and titre of thevirus.

One of skill in the art can determine the appropriate amount based onthe above factors. Vectors may be administered initially in a suitableamount that may be adjusted as required, depending on the clinicalresponse of the patient. The effective amount of a vector can bedetermined empirically and depends on the maximal amount of the vectorthat can be safely administered. In some embodiments, the vector mayhave little cytotoxicity in vertebrates and may be administered in largeamounts. However, the amount of vectors administered should be theminimal amount that produces the desired result.

In various embodiments, a dose of about 10⁹ recombiant baculovirusparticles are administered to a human patient. In other embodiments,about 10² to about 10⁹ recombinant baculovirus particles, about 10⁶ toabout 10⁹ recombinant baculovirus particles, about 10² to about 10⁷recombinant baculovirus particles, about 10³ to about 10⁶ recombinantbaculovirus particles, or about 10⁴ to about 10⁵ recombinant baculovirusparticles may be administered in a single dose. In some embodiments, thevector may be administered more than once, for example, by repeatedinjections. In other embodiments, the viral vector may be repeatedlyadministered though an intrathecal catheter connected to a reservoircontaining a composition comprising the vector, as described in (Jacksonet al. 2001, Human Gene Therapy 12: 1827).

In other embodiments, a non-viral vector comprising about 4 μg of DNAmay be administered to a host in a single dose. Non-viral vectors mayonly transiently express a therapeutic gene once transfected into targetcells, resulting in less than optimal transgene expression. In someembodiments of the invention, the vector may be administered more thanonce, for example, by repeated injection. In other embodiments, thenon-viral vector may be repeatedly administered though an intrathecalcatheter connected to a reservoir containing a composition comprisingthe vector, as described in Jackson et al. 2001, Human Gene Therapy 12:1827).

Transected peripheral nerves may advantageously be treated as follows.The vector encoding a therapeutic gene product, for example, a nervegrowth factor such as NGF, is intrathecally administered to DRG neuronalcells of a host whose transected distal and proximal nerve stumps havebeen secured to a nerve guide conduit. The vector may be administeredinto the cerebrospinal fluid surrounding a dorsal root ganglion todeliver the nucleic acid into the cell body of the transected nerve. Forexample, if the sciatic nerve is transected, the vector may beintrathecally injected by lumbar injection into the cerebrospinal fluidin an intraspine space between the L4 and L5 vertebrae.

As used herein, “nerve guide conduit” also known as a “nerve guidancechannel” or simply “nerve guide” refers to a device with a first andsecond open end connected by an internal passage, wherein the internaldiameter of the conduit is sufficient to accept sections at both ends.Nerve guide conduits may serve to direct axons sprouting from theproximal nerve end, may provide a conduit for the diffusion of growthfactors secreted by the injured nerve ends, and may reduce theinfiltration of scar tissue (Schmidt et al. 2003, Annu Rev Biomed Eng 5:293). As is known in the art, the nerve guide conduit may be derivedfrom biomaterials, such as extracellular matrix proteins, for examplecollagen (U.S. Pat. No. 50,190,987), laminin, fibronectin,fibrin/fibrinogen, hyaluronic acid-bases materials or from materialssuch as, for example silicon, expanded poly(tetrafluoroethylene).Synthetic nerve guide conduits may also be made of biosorbable orbiodegradable materials, for example, such as poly(lactic acid) (PLA),poly(glycolic acid) (PLG), poly (lactic-co-glycolic acid ) (PLGA),poly(caprolactone), poly(urethane), poly(organo)phosphazene,poly(3-hydroxybutyrate) and methacrylate-based hydrogels. (See Schmidt &Leach and reference therein). The nerve guide conduit may be non-porous,porous or semi-porous, may incorporate neuron support cells, such as,for example, Schwann cells, and may have an oriented nerve substratumand may have one or more intraluminal channels (Hudson et al. 1999, ClinPlast Surg 26:617).

A person skilled in the art would know how to secure a transected nerveinto a nerve guide conduit, for example, following Schmidt et al. 2003,Ann. Rev. Biomed. Eng. 293. Generally, the proximal and distal stumps ofa transected peripheral nerve are secured within an internal channel ofa nerve guide conduit by, for example, 9-0 or 10-0 sutures. Preferably,the internal channel of the nerve guide conduit is filled with asolution, suspension or gel supportive of axonal outgrowth.

In normal circumstances, NGF is present very low concentrations, andrapidly increases upon nerve injury in an animal model. Upontransection, NGF is produced mainly by the target tissue and Schwanncells in the distal stump of the damaged nerves, and its retrogradelytransported to the nerve cell body. The exogenous expression of NGFwithin the cell body of neurons in a dorsal root ganglion may supplementor substitute NGF delivered by retrograde transport. Without beinglimited to any particular theory, the exogenous expression of NGF maypromote the anterograde transport of neurotrophic factors to theproximal stump of the transected nerve, and these factors may diffusethough the nerve guide conduit and promote nerve regeneration within theconduit.

As would be understood by a person skilled in the art, theadministration of the vector may be accomplished before, or morepreferably after, the nerve stumps are secured into the conduit. Thetiming of the administration of the vector to the host effective topromote peripheral nerve regeneration can be readily determined by theskilled person.

All documents referred to herein are fully incorporated by reference.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. All technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art of this invention, unlessdefined otherwise.

The word “comprising” is used as an open-ended term, substantiallyequivalent to the phrase “including, but not limited to”. Singulararticles such as “a” and “the” in the specification incorporate, unlessthe context dictates otherwise, both the singular and the plural.

The following examples are illustrative of various aspects of theinvention, and do not limit the broad aspects of the invention asdisclosed herein.

EXAMPLES

Material and Methods

Gene Delivery Vectors

Three non-viral gene delivery systems, polyethyleneimine (PEI)/plasmidDNA complexes, lipofectamine™ 2000/DNA complexes and a peptide-basedsystem, and two viral vectors, recombinant baculovirus andadeno-associated virus Type 2 (AAV-2) vectors, were tested

To prepare PEI/DNA complexes, plasmid DNA pCMV E/P-luc (Liu et al. 2004,Gene Ther. 11:52) or pcDNA3.1/NGF was mixed with PEI (25 kDa;Sigma-Aldrich, San Diego, Calif.) in a 5% glucose solution by adding anappropriate amount of a PEI solution into a DNA solution, briefly mixingby vortexing and waiting for 30 min at room temperature. pcDNA3.1/NGFwas constructed by inserting a full length DNA fragment obtained from amouse brain cDNA library into EcoR1 digested pcDNA3 (Invitrogen). Ratiosof PEI to DNA used for cell transfection and animal experiments were 10and 14 equivalents of PEI nitrogen per DNA phosphate, respectively. Insome experiments, PEI was labeled with the carbocyanine dyes Cy3(Amersham, Uppsala, Sweden) before being mixed with DNA. The requiredamount of the plasmid was calculated by taking into account that 1 μg ofDNA contains 3 nmol of phosphate. To prepare lipofectamine™ 2000/DNAcomplexes, pCMV E/P-luc was mixed with lipofectamine™ 2000 (Invitrogen,Singapore) in a 5% glucose solution by adding the appropriate amount oflipofectamine™ 2000 into the diluted DNA solution, gently mixing andincubating for 20 min at room temperature. Ratios of DNA (in μg) tolipofectamine™ 2000 (in μl) used for animal experiments were 1 to 3.

To prepare a peptide-based gene delivery system, the peptide NL4-10K andPEI600 were used. The peptide contains a 29-amino acid fragment derivedfrom NGF loop 4-containing region (aa80-108) linked with 10-lysineresidue as a DNA binding domain (Zeng et al., J Gene Medicine, 2004, inpress). To prepare DNA complexes, DNA was first complexed with PEI600 ata nitrogen/phosphate ratio of 5 and the peptide was added afterward at apeptide/DNA (nmol/g) ratio of 1.5.

Recombinant baculovirus vectors were constructed according to the manualof Bac-To-Bac® Baculovirus Expression system (Gibco BRL, LifeTechnologies, USA). Luciferase cDNA under control of the human CMV E/Ppromoter or a CMV E/PDGF promoter was from vectors constructed in Liu etal. (Gene Therapy, 2004, 11: 52). The promoter was inserted between NotIand XbaI sites of pFastBac1 and the luciferase cDNA was between XhoI andHindIII sites downstream of the promoter. Recombinant baculorviruseswere propagated in Sf9 insect cells. Budded viruses from insect cellculture medium were filtered through a 0.2-μm pore size filter andconcentrated by ultracentrifugation at 25,000 g for 60 min. For uptakeexperiments, baculoviruses were labeled with the carbocyanine dye Cy3according to the manual provided by the supplier (Amersham, Uppsala,Sweden). Recombinant AAV-2 vectors were constructed by sub-cloning, theCMV E/P promoter from pCMV E/P vector or the CMV E/PDGF promoter frompCMV E/PDGF vector (Liu et al. 2004, Gene Therapy, 11: 52) into amodified pAAV plasmid that is flanked by the ITR sequences. This pAAVplasmid, named as pAAV-MCS-luc, was constructed by replacing theoriginal sequence between the two Not I sites of plasmid pAAV-MCS(Stratagene, La Jolla, Calif.) by a multiple cloning site(MCS)-luciferase-polyA expression cassette that was PCR amplified frompGL3-basic plasmid (Promega, USA) with forward primer,5-ATTGCGGCCGCGGTACCGAGCTCTTACG [SEQ ID NO:1] and reverse primer,5-ATTGCGGCCGCTTATCGATTTTACCACATTTG [SEQ ID NO:2]. The promoter wasinserted into pAAV-MCS-luc between the Kpn I and Hind III sites. Theplasmid was used, together with AAV-2 packaging plasmid pAAV-RC andadenovirus helper plasmid pHelper (Stratagene, La Jolla, Calif.), totransfect HEK293 (human embryonic kidney) cells. AAV-2 vectors that hadbeen packaged in the transfected HEK293 cells were released fromcollected cells by two rounds of freeze/thaw cycles and purified by asingle-step gravity-flow heparin affinity column.

Animals and Operations

Adult male Wistar rats, weighing 250-320 g and supplied by theLaboratory Animal Center, National University of Singapore, were usedthroughout the study. For luciferase activity assays, 50 rats were used,among which 30 rats in the time course study (5 per each time interval),5 each to test PEI, lipofectamine™ 2000, AAV and baculovirus vectors.For immunohistochemical study, 12 rats were used, 6 of which in the shamoperated group and 6 in the experimental group. For PCR analysis, 12rats were used, 6 of which in the sham operated group and 6 in theexperimental group. Rats were randomly assigned to each group at varioustimes after injection. They were kept 4 per cage in a light-dark cycle(12 h/12 h) at a constant temperature of 22° C. and at 60% humidity, andfed with normal laboratory rat food. In the handling and care of allanimals, the International Guiding Principles for Animal Research asstipulated by World Health Organization (1985) and as adopted by theLaboratory Animal Center, National University of Singapore, werefollowed.

For intrathecal injection, rats were anaesthetized by an intraperitonealinjection of sodium pentobarbital (60 mg/kg of body weight). The backskin of rats was incised and the spinal column was exposed. Theintraspine space between lumbar vertebrae 4 and 5 (L4-5) was chosen asthe injection site. A 10 μl micro-syringe connected with a 26-gaugeneedle was used to perform the injection. The slight movement of rattails indicated the proper injection into the subarachnoid space.Complexes with 4 μg of plasmid DNA, 1×10⁸ particles of the AAV-2, or1×10⁷ particles of baculovirus in 20 μl were used for each injection.After a slow administration over 2-5 min, the needle was allowed toremain in situ for 2 min before being removed. The skin was closed withsurgical clips after the injection.

To produce a rat model for nerve injury and regeneration (Xu et al.2003, Biomaterials 24:2504), right sciatic nerves of anesthetized ratswere exposed through a 2 cm long skin incision. A 7 mm piece of thenerve was removed and then the proximal and distal nerve stumps werepulled 2 mm into each opening of a silicone nerve guide conduit (NGC,Tygon® ID: 0.05 inch, OD: 0.09 inch, length: 1.4 cm), leaving a 10 mminterstump gap. Twenty-five micro liter of saline was filled into thetube before the proximal stump was pulled into the tube opening. The twostumps were fixed to the tubes with a single 10/0 perineurial suture(Ethilon). The intrathecal injection into the intraspinearea between L4and L5 vertebrae of 20 μl of PEI complexes containing 4 μg of pcDNA/NGF,or PEI/pcDNA3/luc complexes for control rats, was carried on immediatelyafter NGC implantation Thirty rats were used in this experiment, 15 ofwhich received the injection of PEI/pcDNA3.1/NGF complexes and the other15 rats as controls.

Reporter Gene Detection

PCR amplification was carried out to detect transported reporter genesin DRG. 2 days after lumbar intrathecal injection of PEI/DNA complexesor baculovirus vectors, rats were sacrificed by intracardiac perfusionwith 0.1 M PBS (pH 7.4) following deep anesthesia and three pairs ofcompanion DRGs (lumbar L4 to L6) around the injection site werecollected. The tissues were homogenized by mincing with a razor bladeand DNA was extracted according to the standard protocol of a DNeasyTissue Kit (Qiagen, Hilden, Germany). Oligonucleotide primers for PCRwere designed based on the luciferase gene sequence and are listedbelow: 5′ primer, 5′-AT TGC TCA ACA GTA TGG GCA-3′ [SEQ ID NO:3], 3′primer: 5′-CGA AGA AGG AGA ATA GGG TTG-3′ [SEQ ID NO:4]. The expectedsize of the amplified product is 540 bp. Amplification cycles consistedof 94° C. 5 min, 1 cycle; 94° C., 45 s, 55° C. 30 s, 72° C., 30 s, 35cycles with a final extension at 72° C. for 7 min. To excludecontamination of the whole process, samples from sham-operated rats werehandled in parallel.

The expression of the luciferase reporter gene was examined using aluciferase activity assay. Rats were sacrificed at 2 days post-injectionby intracardiac perfusion with 0.1 M PBS (pH 7.4) following deepanesthesia. Six pairs of companion DRG around the injection site werecollected and stored at −80° C. until processing. After adding PBSbuffer (100 μl PBS per 50 mg tissue), each sample was homogenized bysonication for 10 seconds on ice, and then centrifuged at 13,000 rpm at4° C. in a microcentrifuge. Ten microliters of the supernatant at roomtemperature was used for the luciferase activity assay employing anassay kit from Promega (Madison, Wis., USA). Measurements were made in asingle-well luminometer (Berthold Lumat L B 9501) for 10 seconds. RLUswere normalized by the total protein concentration of the tissueextracts, measured with a protein assay kit (Bio-Rad, Hercules, Calif.,USA).

For immunostaining, rats were sacrificed at 2 days post-injection.Following deep anesthesia, rats were perfused first with Ringer'ssolution followed by 2% paraformaldehyde in 0.1 M PBS (pH 7.4). Afterperfusion, 3 pairs of companion DRGs (L4 to L6) were removed andpost-fixed in the same fixative for 2-4 hours before being transferredinto 0.1 M PBS containing 15% sucrose. Frozen sections were cut at 30 μmthickness and mounted on coated slides. Sections were washed for 20 minin 0.1M PBS at pH 7.4 containing 0.2% Triton X-100, then blocked with 5%normal goat serum in PBS for 1 hour. Sections were then incubatedovernight with primary antibodies polyclonal anti-luciferase (Promega;dilution 1:150) and monoclonal against neuron-specific nuclear protein(NeuN) (Chemicon International, USA; dilution 1:500). Sections werewashed in 0.1 M PBS and further incubated with anti-rabbit IgG Tritcconjugate (Sigma-Aldrich, Inc., USA; dilution 1:100) and anti-mouse IgGFitc conjugate (Sigma-Aldrich; dilution 1:100) for 1 hour. Afterincubation, sections were washed three times in PBS, mounted with DAKOfluorescent mounting medium and covered with coverslips. Controlsections were incubated without primary antibodies. Sections wereexamined with an Olympus 500 confocal laser scanning microscope. Eachsection was initially scanned with a 488 nm laser line, and an emissionfilter BP 510-525, for the detection of Fitc fluorescein; and with a 543nm laser line, and an emission filter LP 570, for the detection of Tritcfluorescein.

NGF Assay

Cos7 cells were used for in vitro transfection to test the geneexpression from the plasmid pcDNA3.1/NGF. Cultured Cells were seeded ina 6-well plated at 60-70% confluency. After overnight incubation, theculture medium was replaced with Opti-MEM and an aliquot of 25 μl ofPEI/DNA complexes containing 4 μg pcDNA3.1/NGF was added to each well.DNA/PEI complexes were incubated with the cells for 3 hours at 37° C.The medium was then replaced with fresh complete medium. After a furtherincubation for 24 hours, the cells and the medium were collected for NGFassays using a sensitive NGF ELISA Kit (Boehringer Mannheim). For thecontrol group, pcDNA3-Luc was used instead of pcDNA3.1/NGF. For In vivoNGF expression analysis, 15 rats were used, 10 of which were used in theexperimental group, 5 for each time interval at 3 and 7 days. The left 5rats were used as normal controls. Four μg of pcDNA3.1/NGF or pcDNA3lucwas complexed with PEI in 20 μl and injected into each rat as describedabove. DGR were collected and homogenised. The supernatants were usedfor NGF ELISA.

Evaluation of Nerve Regeneration

Four weeks post-operation, rats were anaesthetized again and the sciaticnerves together with NGCs were exposed and carefully isolated from thesurrounding tissues. The nerve segment distal to the tube was pinchedwith a pair of forceps to identify the success of the nerveregeneration. Contraction of muscles on the back or retraction of theleg indicated the presence of regenerating sensory fibers in thepinching segment, while no response was taken as an indication of theabsence of such fibers.

For histological examination, regenerated nerve cables within the NGCswere collected and fixed in 2.5% glutaradelyde in PBS buffer (pH=7.4)overnight. The subsequent fixation, embedding, sectioning, and stainingprocedures were the same as previously described (Xu et al. 2003,Biomaterials 24:2504). The distal segments of the regenerated nerve werethen performed with semi-thin section and stained with Tolubine Blue formorphometric analysis. Seventeen transverse semi-thin section samplesthrough the middle part of the 10-mm gaps were analyzed to determine thenumber of regenerated axons, fiber population and fiber diameter.Quantitative measurement and evaluation was carried out using MicroImage Lite™ (Olympus, Image Analysis Software). Areas of interest wereselected for ultrathin sectioning. The ultrathin sections (100 nm) werestained with lead citrate, collected on copper mesh grids and examinedin Philips EM 208s electron microscope operating at 80-100 kV. Thesamples were evaluated for regeneration of nerve tissue and foreign bodyreaction against the polymers

Example 1 Gene Transfer to DRG via Lumbar Intrathecal Injection

We started with evaluating the uptake of gene vectors by DRG afterintrathecal administration of baculovirus vectors or PEI/DNA complexescovalently labelled with Cy3. DRG close to the injection site werecollected at 2 days post-injection and their sections were stained byanit-NeuN. Red Cy3 signals were detectable in the DRG, mainly in thecytoplasm of NeuN-positive cells under higher magnifications (FIG. 1);PCR analysis of the samples of DRG collected at day 1 and 3post-injection revealed the existence of transported reporter genes inthese PNS regions after the CNS injection (FIG. 2).

Four different types of gene delivery systems and vectors with aluciferase reporter gene, PEI/DNA complexes, lipofectamine/DNAcomplexes, baculovirus vectors and AAV-2 vectors, were tested for theireffects in mediating transgene transfer into DRG cells. Two days afterlumbar intrathecal injection, luciferase activities were easily detectedin DRG cells for all types of the vectors (FIG. 3). In a time coursestudy of transgenic luciferase expression from the PEI/DNA complexes,enzymatic activity could be detected as early as 1 day after injection,reached the peak at day 3, and then dropped over next several weeks,with a low activity still being detectable 4 weeks after injection (FIG.3). The immunostaining of luciferase demonstrated that the proteindetected in DRG was not limited on the epithelium, but also in ganglioncells. The double immunostaining for luciferase and the neuron-specificNeuN protein showed that most of the luciferase-positive cells in DRGswere also NeuN-positive, with only few luciferase-positive cells beingnon-neuronal cells (FIG. 5).

Example 2 NGF cDNA Transfection and Nerve Regeneration through NerveGuide Conduits

NGF, a neurotrophic factor predominantly acting on sensory andsympathetic neurons (Thorne, R. G., and Frey, W. H. II 2001, ClinPharmocokinet 40:907), was selected to test the effects of itsexpression in DRG on peripheral nerve regeneration through nerve guideconduits (NGCs), a device widely tested in pre-clinical studies torepair nerve defects (Schmidt et al. 2003, Ann. Rev. Biomed. Eng. 293).We examined the expression of NGF cDNA from PEI-mediated gene delivery.After transfection of COS7 cells, the NGF concentration increased in theculture media, as detected in a sensitive ELISA, with a level of about 1ng per ml and being 6 folds higher than the control (FIG. 6A). Similarincrease was also observed when the cell lysate was analysed (FIG. 6B).After lumbar intrathecal injection of PEI/NGF cDNA complexes, theexpression level of NGF in DRG was three-fold higher than the controland persisted for at least 7 days.

Example 3 Effects of NGF Expression on Nerve Regeneration

The successful rate of nerve regeneration through NGCs, as demonstratedin a pinch test, was 87% in the NGF group at 4 weeks post-operation,versus 67% in the control group. The regenerated tissue cables, whichhad bridged a 10-mm gap between two nerve stumps, could be found insidethe conduits collected from the rats that were positive in the pinchtest. Those tissue cables collected from NGCs of the NGF group showedimproved regeneration qualities over their controls, with significantincrease in the diameter of nerve fibers and decrease in G-ratio(Friende et al. 1982, Brain Research 235:335), a ratio determined byaxon diameter vs. myelinated fiber diameter (FIG. 7). In other twoparameters examined, fiber population and density, ever though thecertain improvement in the NGF group was visible, no statisticallysignificant difference was found, probably due the big standarddeviation (FIG. 7).

Morphological hallmarks of nerve regeneration were also examined bytransmission electron microscopy. The regenerated nerve cables werecentrally located within the conduits, surrounded by a fine epineurium.In both the NGF and control groups, the cables contained numerous offascicles of regenerated myelinated as well as unmyelinated axons. Thenumber, diameter and density of myelinated axons in the NGF group weremore, lager and higher than those in the control group, respectively(FIG. 8). The myelin sheath presented in the NGF group was also muchthicker than that in the control group (FIG. 8).

The present study thus demonstrates the positive influence oftherapeutic NGF gene on the regeneration of transected sciatic nerves,providing one example of gene therapy for peripheral nerve regeneration.NGF is the first and best-characterized nerve-derived factor and acts ona relatively limited variety of neuronal population, includingsympathetic and subpopulations of sensory neurons of peripheral nervoussystem, striatial and septal cholinergic neurons in the brain (Terenghi,G. 1999, J. Anat. 184:1). NGF is present at a very low concentration inthe normal circumstances, and rapidly increases in the experimentalnerve injury animal model. The proteins are produced mainly by thetarget tissue and Schwann cells in the distal stump of damaged nervesand then transported in a retrograde manner to the cell soma beforeacting on receptors on neurons and producing the neurotrophic effects.With peripheral neuropathy, such transport within diseased nerves may benegatively reduced or even totally blocked. An alternative means tomaintain the transport mechanism thus uphold normal functions ofaffected neurons would be essential in treatment of peripheralneuropathy. Gene vectors delivered to DRG neurons by intrathecalinjection do not require axonal transport to reach the cell body andcould be beneficially utilized to support functions of neurons in DRG,thus slowing down or stopping degeneration processes of concerned axons.

NGF transfection in DRG, nerve regeneration, especially the diameter ofnew axons, was improved as demonstrated by both of the morphometricanalysis and TEM analysis. The number of neurofilaments within axons hasbeen considered as the key factor to control axonal diameter and insensory neurons is probably subject to the regulation of NGF. Followingperipheral nerve transection, nerve body size, axon diameter,neurofilament synthesis, and axonal transport of neurofilament are allreduced in sensory neurons. The transfection of NGF cDNA wouldup-regulate the expression of NGF protein and might have contributed tothe enhancement of neurofilament synthesis and significant improvementin fiber diameter observed in the current study.

As can be understood by one skilled in the art, many modifications tothe exemplary embodiments described herein are possible. The invention,rather, is intended to encompass all such modification within its scope,as defined by the claims.

1. A method of delivering a nucleic acid into a neuronal cell in theperipheral nervous system of a host comprising the step of identifying atarget neuronal cell in a dorsal root ganglion and administering avector comprising the nucleic acid into a site in the cerebrospinalfluid of the host wherein the site is sufficiently proximal to thedorsal root ganglion to deliver the nucleic acid into the cell body ofthe target neuronal cell.
 2. A method of treating a peripheralneuropathy in a host, the method comprising identifying a targetneuronal cell in a dorsal root ganglion affected by the neuropathy, andadministering a vector comprising a therapeutic nucleic acid into a sitein the cerebrospinal fluid of the host wherein the site is sufficientlyproximal to the dorsal root ganglion to deliver the nucleic acid intothe cell body of the target neuronal cell.
 3. A method of treating atransected peripheral nerve having a proximal and a distal stump in ahost, the method comprising administering a vector comprising atherapeutic nucleic acid to a dorsal root ganglion neuronal cell whereinthe distal and proximal stumps are secured to a nerve guide conduit. 4.The method of claim 3 wherein administering the vector to a dorsal rootganglion neuronal cell comprises administering the vector at a site inthe cerebrospinal fluid sufficiently proximal to the dorsal rootganglion neuronal cell to deliver the nucleic acid into the cell body ofthe dorsal root ganglion neuronal cell.
 5. The method of claim 2 whereinthe peripheral neuropathy is diabetic neuropathy, nerve compression ornerve transection.
 6. The method of claim 1 wherein the nucleic acidcomprises a coding sequence operably linked to a promoter.
 7. The methodof claim 6 wherein the promoter is a viral promoter.
 8. The method ofclaim 7 wherein the viral promoter is a CMV promoter.
 9. The method ofclaim 6 wherein the promoter is a neuron-specific promoter.
 10. Themethod of claim 9 wherein the neuron-specific promoter is a PDGF βpromoter.
 11. The method of claim 6 wherein the promoter is operablylinked to an enhancer.
 12. The method of claim 11 wherein the enhanceris a CMV enhancer.
 13. The method of claim 2 to wherein the nucleic acidencodes a neurotrophic factor.
 14. The method of claim 13 wherein theneurotrophic factor is a nerve growth factor.
 15. The method of claim 14wherein the nerve growth factor is NGF.
 16. The method of claim 2wherein the nucleic acid encodes an anti-apoptotic factor.
 17. Themethod of claim 16 wherein the anti-apoptotic factor is bcl-2.
 18. Themethod of claim 1 wherein the vector is a viral vector.
 19. The methodof claim 18 wherein the viral vector is a baculoviral vector or an AAVvector.
 20. The method of claim 1 wherein the vector is a non-viralvector.
 21. The method of claim 20 wherein the non-viral vector is apolymer-based vector, a polypeptide-based vector or a lipid-basedvector.
 22. The method of claim 21 wherein the polymer-based vector ispolyethyleneimine.
 23. The method of claim 1 wherein the vector isadministered by injection.
 24. The method of claim 23 wherein the vectoris administered by lumbar injection. 25-47. (canceled)